Separation and partial characterization of two alkaline peptidases from cotyledons of resting kidney beans, Phaseolus vulagaris

Extracts prepared from cotyledons of resting kidney beans ( Phaseolus vulgaris L., cv. Processor) rapidly hydrolyzed two dipeptides, Leu-Tyr and Ala-Gly, with pH optima at 9.2 and 8.5, respectively. On ion exchange chromatography on DEAE-Sephacel the two activities eluted as separate peaks, showing that they were due to two different peptidases. The extracts also hydrolyzed Leu-β-naphthylamide optimally at pH 6.4; this activity eluted as a third peak berween the other peaks. The activity peak acting on Leu-Tyr and Ala-Gly rapidly hydrolized two tripeptides, showing that it was an aminopeptidase, whereas the Ala-Gly hydrolyzing peak acted only on dipeptides. The activities against Leu-Tyr and Ala-Gly were also separated by gel chromatography on Sephacryl S-300 with elution positions corresponding to M, values of about 360 000 and 105 000. The aminopeptidase was inhibited by bestatin, and the dipeptidase was inhibited by p-hydroxymercuribenzoate. Both enzymes were inhibited by o-phenanthroline. In most of their properties the two kidneys bean enzymes resembled the alkaline aminopeptidase and the dipeptidase earlier characterized from barley grains.     

Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris

The activities of aminopeptidase (EC 3.4.11), dipeptidase (EC 3.4.13), carboxypeptidase (EC 3.4.16), naphthylamidase (EC 3.4.11) and proteinases (EC 3.4.21) were assayed in extracts from the cotyledons and the axial tissues of resting and germinating kidney beans ( Phaseolus vulgaris L. cv. Processor).
The activities of the alkaline peptidases (aminopeptidase hydrolyzing Leu-Tyr at pH 9.2 and dipeptidase acting on Ala-Gly at pH 8.5) and naphthylamidases (hydrolyzing Leu-β-naphthylamide at pH 6.4) were high in the cotyledons of resting seeds, but decreased during germination. This decrease was faster than the loss of the total nitrogen. On the contrary, the activities of carboxypeptidase (hydrolyzing carbobenzoxy-Phe-Ala at pH 5.9) and proteinases (acting on haemoglobin at pH 3.7 and on casein at pH 5.4 and 7.0) were low in the resting seeds, but increased during germination reaching their maximal values when the mobilization of nitrogen was highest. It has been suggested that the breakdown of storage proteins is initiated inside the protein bodies by acid proteinases and carboxypeptidases. Although the activities of the alkaline peptidases and naphthylamidases decreased during germination, these were still relatively high and enough for the completion of the proteolytic breakdown. Thus, it is suggested that, as a final step in a chain of events, the main role for the alkaline peptidases in the cotyledons of germinating seeds is to provide amino acids for the growth of the seedling.     

Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

A leucine aminopeptidase (EC 3,4,11.1) was purified from cotyledons of resting kidney beans ( Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M, of 360 000. The enzyme gave one band on native gel electrophoresis and an electrophoretic titration in an immobilized pH gradient gave a single curve with a pI of 4.8. Two bands were observed in an SDS-gel electrophoresis with M_r values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disulphide bridges. The purified enzyme most rapidly liberated Leu and Ala of the N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The enzyme was stable in the presence of glycerol, dithiothreitol and Mg²⁺, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kⁱ-value of 1.5 nM . These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4, 11.1). The high enzyme activity observed suggests that this aminopeptidase has an important role in the production of free amino acids during germination.     

Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a K(m) of 4.26x10(-5)m for RS-mevalonate and 1.54x10(-3)m for ATP. The preparation from green leaves had a K(m) of 4.55x10(-5)m for RS-mevalonate and 1.75x10(-3)m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.     

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean, Phaseolus vulgaris

8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins.     

Intracellular localization of CA1P and CA1P phosphatase activity in leaves of Phaseolus vulgaris L.

CA1P and CA1P phosphatase occur in the chloroplasts of leaf mesophyll cells of many species. However, whether either may occur exclusively in the chloroplast has not yet been established. To examine their intracellular distribution, mature, dark-or light-treated leaves of Phaseolus vulgaris were frozen, lyophilized and then centrifuged in density gradients of heptane and tetrachloroethylene. After gradient fractionation, both CA1P and CA1P phosphatase activity co-segregated with chloroplast material. Distribution analyses using sub-cellular compartment markers indicated that both CA1P and CA1P phosphatase do occur exclusively in leaf chloroplasts.Abbreviations Bicine N,N-bis[2-hydroxyethyl]glycine - CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - Chl chlorophyll - DTT dithiothreitol - EDTA (ethylenediamine)tetraacetic acid - PEP phosphoenolpyruvate - Tris tris(hydroxymethyl)aminomethane     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

Purification and properties of phaseolamin, an inhibitor of alpha-amylase, from the kidney bean, Phaseolus vulgaris.

Kidney beans, Phaseolus vulgaris, contain a proteinaceous inhibitor of alpha-amylase, which we have named phaseolamin. The inhibitor has been purified to homogeneity by conventional protein fractionation methods involving heat treatment, dialysis, and chromatography on DEAE-cellulose, Sephadex G-100, and CM-cellulose. Phaseolamin is specific for animal alpha-amylases, having no activity towards the corresponding plant, bacterial, and fungal enzymes, or any other hydrolytic enzyme tested. Optimal inhibitory activity is expressed during preincubation of enzyme and inhibitor at pH 5.5 and 37 degrees. Substrate prevents inhibition. Measurement of the stoichiometry on inhibition showed that a 1:1 complex of alpha-amylase and inhibitor is formed. Complex formation was demonstrated by chromatography on Sephadex G-100. The phaseolamin-amylase complex is dissociated at low pH values, apparently as a result of destruction of the enzyme; the complex cannot be dissociated by other conditions unfavorable for inhibition (low temperature or high pH). Phaseolamin inhibits hog pancreatic alpha-amylase in a noncompetitive manner.     

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.Abbreviations Rubisco ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) - LSU large subunit of Rubisco - SSU small subunit of Rubisco - ABA abscisic acid - FW fresh weight     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

A study of the subcellular localisation of an ethylene binding site in developing cotyledons of Phaseolus vulgaris L. by high resolution autoradiography

Use was made of light microscopy and high resolution electron microscope autoradiography to determine the subcellular localisation of a binding site with a high affinity and specificity for ethylene in developing cotyledons of Phaseolus vulgaris L. The results indicate that the binding site is located on the endoplasmic reticulum and protein body membranes, confirming previous studies using cellular fractionation and marker enzymes.     

Regulation of cell-wall polysaccharide components by CaCl2 in suspension cultures of kidney bean (Phaseolus vulgaris)

We cultured the suspension cells of kidney bean in MS media supplemented with one of five concentrations of CaCI₂ [0,22,44 (control), 88, or 176 mg/L], and harvested them at the logistic (15 d) and early-stationary (30 d) phases. Cells grown at concentrations higher than 22 mg/L showed better proliferation than those at 0 mg/L The rate of proliferation also increased with higher concentrations. We fractionated the individual sugars into symplastic (EtOH and starch) and apoplastic (low-molecular pectin, high-molecular pectin, hemicellulose, and cellulose) components. Cells treated at the highest concentration (176 mg/L) exhibited the greatest amount of sugar in the EtOH and starch fraction during the logistic phase. In contrast, cells in the early stationary phase had the highest level of sugar at treatment concentrations of less than 22 mg/L. For treatment concentrations higher than 22 mg/L on Day 15, more pectin and hemicellulose was detected at greater amounts compared with those cells treated with 0 mg/L. However, at Day 30, concentrations higher than 44 mg/L induced greater amounts of pectin and hemicellulose than from the other concentrations. Cellulose was more abundant with the 0 mg/L treatment, and contents ranged from 17.4 to 25.5% in the primary cell walls over all treatment concentrations. These results indicate that CaCI₂ modulates both symplastic and apoplastic sugar metabolism. Therefore, we suggest that the cell-wall structure may define the mode of polysaccharide biosynthesis during cell growth.     

Effects of elevated temperature and carbon dioxide on seed-set and yield of kidney bean (Phaseolus vulgaris L.)

It is important to quantify and understand the consequences of elevated temperature and carbon dioxide (CO₂) on reproductive processes and yield to develop suitable agronomic or genetic management for future climates. The objectives of this research work were (a) to quantify the effects of elevated temperature and CO₂ on photosynthesis, pollen production, pollen viability, seed‐set, seed number, seeds per pod, seed size, seed yield and dry matter production of kidney bean and (b) to determine if deleterious effects of high temperature on reproductive processes and yield could be compensated by enhanced photosynthesis at elevated CO₂ levels. Red kidney bean cv. Montcalm was grown in controlled environments at day/night temperatures ranging from 28/18 to 40/30 °C under ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ levels. There were strong negative relations between temperature over a range of 28/18–40/30 °C and seed‐set (slope, ? 6.5% °C^?1) and seed number per pod (? 0.34 °C^?1) under both ambient and elevated CO₂ levels. Exposure to temperature > 28/18 °C also reduced photosynthesis (? 0.3 and ? 0.9 µmol m^?2 s^?1 °C^?1), seed number (? 2.3 and ? 3.3 °C^?1) and seed yield (? 1.1 and ? 1.5 g plant^?1 °C^?1), at both the CO₂ levels (ambient and elevated, respectively). Reduced seed‐set and seed number at high temperatures was primarily owing to decreased pollen production and pollen viability. Elevated CO₂ did not affect seed size but temperature > 31/21 °C linearly reduced seed size by 0.07 g °C^?1. Elevated CO₂ increased photosynthesis and seed yield by approximately 50 and 24%, respectively. There was no beneficial interaction of CO₂ and temperature, and CO₂ enrichment did not offset the negative effects of high temperatures on reproductive processes and yield. In conclusion, even with beneficial effects of CO₂ enrichment, yield losses owing to high temperature (> 34/24 °C) are likely to occur, particularly if high temperatures coincide with sensitive stages of reproductive development.     

The isolation of two proteins, glycoprotein I and a trypsin inhibitor, from the seeds of kidney bean (Phaseolus vulgaris)

1. The isolation of two proteins from the seeds of kidney bean is described. 2. The individual steps in the purification procedure included: extraction of the seeds at pH9.0, dialysis, first against pH9.0 and then against pH5.0 buffers, high-voltage electrophoresis of the proteins soluble at pH5.0 and chromatography on Sephadex G-200, Sephadex G-75 and DEAE-Sephadex columns. 3. Of the two proteins isolated, the first and larger component was a glycoprotein and its carbohydrate part was mainly composed of d-mannose and d-glucosamine together with smaller amounts of arabinose, xylose and fucose. 4. The second protein component isolated was a trypsin inhibitor and was almost entirely devoid of sugars but contained a firmly bound pinkish-blue pigment. 5. The amino acid composition of the two proteins was determined. 6. The glycoprotein contained very little if any cyst(e)ine but was relatively rich in aromatic amino acids, whereas the trypsin inhibitor had an unusually high cystine content (nearly 15%) but was relatively poor in valine and in aromatic amino acids.     

Changes in proteolytic enzyme activities and transformation of nitrogenous compounds in the germinating seeds of kidney bean (Phaseolus vulgaris)

Summary Hydrolytic activity towards synthetic substrates and denatured proteins was measured in the extracts of the seeds of kidney bean at various stages of germination up to 16 days.Of the peptide hydrolases, chymotrypsin-type activity was stable for the first 7 days, then rapidly increased towards the end; leucine aminopeptidase activity decreased to a minimum (8th day) then slowly increased again; trypsin-type activity remained constant throughout.Proteolytic and autodigesting activities showed an optimum between pH 5.0 and 5.5. Both activities decreased slowly first, then rose to a sharp maximum at the 8th day. The haemoglobin-digesting activity after a minimum increased again at the 14th day. The autodigesting activity had an additional maximum.Concomitant with these changes, non-protein nitrogen increased twofold by the 5th day, remained constant up to the 12th day and then increased again. Protein content on the other hand decreased first, had a maximum at the 9th day after which it steadily decreased again. The amounts of albumins and globulins changed independently of each other: albumins decreased continuously with the exception of a steady period (5–9th days), while globulins were more stable except for a sharp minimum (6–7th days) and a steady decrease after the 13th day.The following abbreviations were used: BAPA, -N-benzoyl-L-arginine-p-nitroanilidine; N-CBZ-Tyr-PA, -N-carbobenzoxy-L-tyrosine-p-nitrophenyl ester; LPA, leucyl-p.nitroanilidine; BAEE, -N-benzoyl-L-arginine ethyl ester, TCA, trichloroacetic acid.     

Transport and redistribution of selenium in the bean plant (Phaseolus vulgaris)

After incubation for 3 h with (⁷⁵Se) selenate, the selenium distribution in the bean plant (Phaseolus vulgaris L. cv. Contender) through a 29-day period showed an uneven distribution: roots and trifoliate leaves were richer in ⁷⁵Se than stem and primary leaves. The high selenium concentration of roots resulted from the retention of selenate by the root cells: at the end of the 29-day period about 60° of the radioactivity was always ethanol-soluble, and when analysed by paper chromatography, proved to be selenate. By contrast, much of the radioactivity of the leaves was ethanol-insoluble, ⁷⁵Se being quickly captured in metabolic processes which immobilize it. During plant development, a portion of the total selenium remains mobile and is continually mobilized to the younger organs which display a rapid growth rate. This delivery results from a progressive liberation of selenate retained by mature organs, especially the roots, and from turnover in older leaf tissues, especially the trifoliate leaves.